1. Field of the Invention
The field of the invention relates to nucleotide factor kappa B promoter and to methods of controlling its activity in connection with a wide range of diseases. The invention provides methods of treating these diseases and provides protocols for identifying individuals who are susceptible to or at increased risk for an adverse response to stress, injury or infection. In particular, the invention relates to inflammatory diseases and to identification of at risk individuals who exhibit a singular polymorphism in the nucleotide factor kappa-B (NFKB) promoter. The polymorphism is functionally related to a high risk of disease.
2. Background
Nuclear Factor-kB
NF-kB transcription factors play an important role in regulating basic cellular functions. Abnormal NF-kB activities have been implicated in numerous disease states such as cancers, AIDS, autoimmunity and neurodegenerative diseases. Studies in mice indicate that lack of both p50 and p105 caused by deletion of the nfkb1 gene does not interfere with normal development but does alter normal immune response to pathological infections (Lin and Kobayashi, 2003).
Nuclear Factor-kB is a major transcription regulator of immune response, apoptosis and cell-growth control genes and is also an important mediator of the chronic inflammation associated with a wide range of diseases and pathological conditions, including cancer, infection, response to biological stressors and, particularly, autoimmune diseases (Baldwin, 2001). Inflammatory Bowel disease (IBD) in particular is thought to be associated with NFKB (Schreiber, 1998). IBD includes Crohn's Disease (CD) and ulcerative colitis (UC).
NF-κB is involved in the expression of several cytokines and adhesion molecules. Cytokines, for example, are produced by immune cells, and some induce proliferation and differentiation of specific cells while others induce an acute phase response in inflammation. Inflammation may be induced by acute phase response proteins such as angiotensinogen, serum amyloid protein, al acid glycoprotein, C3 complement or complement factor B. There is thus an important role of NF-κB modulating cytokine expression at the gene level.
NF-κB is thought to be involved in a wide variety of human diseases, including atherosclerosis, asthma, arthritis, cachexia, cancer, diabetes, euthyroid sick syndrome, AIDS, inflammatory bowel disease and stroke.
In most cells before stimulation, NF-κB primarily resides in the cytoplasm in inactive complexes through association with a sequestering inhibitory protein, termed IκB. A wide range of stimuli, including bacterial and viral products, cytokines and oxidant-free radicals, activate NF-κB. These stimuli promote NF-κB nuclear translocation by a mechanism that involves IκB phosphorylation and the ubiquitin-proteosome pathway. This phosphorylation appears to target IκB for degradation and leads to its dissociation from the NF-κB complex and subsequent translocation of NF-κB to the nucleus. There, active NF-κB binds to genomic DNA at promoter regions and thereby regulates gene transcription.
Inappropriate activation of NF-κB has been implicated in inflammation associated with a variety of human diseases and pathologic conditions, among them asthma, inflammatory arthritis, septic shock, lung fibrosis, diabetes, cancer, AIDS, atherosclerosis, stroke and IBD (Baldwin, 2001). Furthermore, several anti-inflammatory and anti-cancer drugs work in part through inhibition of NF-κB activation. For example, aspirin and glucocorticoids inhibit NF-κB (15,16). Consistent with NF-κB regulation of genes involved in the immune and inflammatory responses, mice null for several of the NF-κB subunits show defects in clearing bacterial infection along with defects in B-cell and T-cell functions.
NF-κB is thought to play a central pathogenic role in chronic intestinal inflammation. Activated NF-κB was increased and found localized to the macrophages and epithelial cells in the inflamed intestinal mucosa of CD and UC patients using immunohistochemistry methods. Schreiber et al. (1998) have found that CD and UC patients have increased NF-κB activity in intestinal lamina propria cells. Additionally, the therapeutic properties of mesalazine and sulfasalazine (the most common specific medical therapies for mild to moderate UC) rely in part on inhibition of NF-κB activation. Three CD associated mutations in the NOD2/CARD 15 gene on chromosome 16 all have a defect in their ability to activate NF-κB. This may cause a defect in the innate immune system's ability to protect the gut against invasive bacteria (Hisamatsu, Suzuki et al., 2003).
Inflammatory Bowel Diseases
Ulcerative colitis (UC) and Crohn's disease (CD) are idiopathic, chronic, frequently disabling, inflammatory bowel diseases (IBD). UC is characterized by mucosal inflammation limited to the colon, always involving the rectum and a variable extent of the more proximal colon in a continuous manner. CD inflammation is transmural, most often discontinuous and may involve any portion of the gastrointestinal tract but most commonly involves the distal ileum. The prevalence of IBD in the United States is 200-300/100,000 with a similar prevalence for UC and CD. IBD is considered a complex genetic disorder involving multiple genes of relatively low penetrance, since the familial patterns of inheritance do not conform to simple Mendelian models. Overall, 10-20% of individuals with IBD report one or more relatives with IBD. Relatives of CD patients have a 10-fold risk of developing CD and relatives of UC patients have an 8-fold risk of developing UC. However, these diseases appear to be genetically related, as relatives of CD patients have a 4-fold risk of developing UC and relatives of UC patients have a 2-fold risk of developing CD.
Ulcerative Colitis (UC)
A genetic contribution to the pathogenesis of UC has remained largely unclear. While genome-wide searches have identified several loci in linkage with the disease, case-control studies have only shown a reproducible association between UC and HLA class II genes, especially DRB1*0103 and DRB1*15 (Brant, Okazaki, 2003). Most studies have focused on HLA class II genes, although there is an increasing interest in the role of cytokines in UC pathogenesis and on the polymorphic genes that may influence cytokine secretion.
NFKB Gene
NFKB1 may be the first of perhaps several modest polymorphic risk genes that are involved with inflammation pathways associated with UC. Other cytokine regulators of the pathway that have been shown to have functional polymorphisms, and that ultimately NF-κB protein activation, include interleukin 1 receptor antagonist (IL1RN) and IκB-like gene (NFKBIL1). There is conflicting evidence for an association of allele 2 of IL1RN, the gene that encodes the interleukin 1 receptor antagonist and preliminary evidence of an association of NFKBIL1 with UC (De la Concha, Femandez-Arquero, et al., 2000). An association of the TNF(−857C) promoter polymorphism with IBD (both CD and UC) has also been reported (van Heel, et al., 2002).
NFKB1 gene, located at chromosome 4q24 is an important candidate gene for inflammatory bowel disease. The encoded Nuclear Factor-κB (NF-κB) proteins are a family of transcription factors that regulate various biological defense processes, most notably innate and adaptive immune responses, acute phase reaction and apoptosis. There are five members of the NF-κB family in mammals: p50/p105, p65/RelA, c-Rel, RelB and p52/p100. Although many dimeric forms of NF-κB have been detected, the major form of NF-κB is a heterodimer of the p50 and p65/RelA subunits, encoded by the genes NFKB1 and NFKB3, respectively. Human NFKB1 encodes two proteins, a 105 kDa, non DNA-binding, cytoplasmic molecule (p105), and a 50 kDa DNA-binding protein (p50) that corresponds to the N-terminus of p105. The NFKB1 gene spans 156 kb and has 24 exons with introns varying between 40 000 and 323 bp in length (FIG. 1).
NFKB1 has also been implicated in numerous inflammatory diseases and risk factors for immune-mediated conditions. An association has been reported between an NFKB1 microsatellite and type I diabetes in one instance. No associations have been found with NFKB1 and the exon 12+77C>T polymorphism for multiple sclerosis or Parkinson's disease (Wintermeyer, Riess, et al., 2002). LD is likely incomplete between the exon 12 SNP and the disclosed −94del/insATTG polymorphism. No functional NFKB1 genetic polymorphisms other than the −94del/insATTG have been described.
Deficiencies in the Art
There is a distinct need to identify and understand the role of genetic factors in the development of human disease, and to identify and treat those at risk for disease. Unfortunately, for many conditions, early detection is not possible so that early stage intervention and treatment opportunities are not available. Identification of a direct functional relation between atypical gene nucleotide sequences in polymorphic promoters and abnormal biological function as manifested in various diseases has yet to be established. Determination and location of the polymorphisms will allow development of diagnostic probes for clinical disorders.
Establishment of a functional relation between a promoter gene polymorphism and a pathology would provide new opportunities for intervention at the most basic stage of disease development. Interventions could be developed based on gene therapies, on alteration of transcription factors or on modification of identified nuclear binding proteins. Clearly, it would be desirable to control abnormal gene function at the gene level rather than far downstream in a damaging cascade of intertwined metabolic cycles.